Sequence of KREPB4 variant library copy constitutively expressed from beta-tubulin locus in Trypanosoma brucei brucei
5 x 107 BF B4 CN cells containing pHD1344tub(NAT-HSVTK)-Sce1CS-SceI-ddDHFR were treated with 100 M trimethoprim for 6 h to induce I-SceI-mediated cleavage of the target locus, then transfected with 12.5 g of NotI-linearized pHD1344tub(PAC)-mutRIIIKREPB4-Cterm3V5 plasmid containing the mutant library using the Amaxa nucleofector. Following transfection, cells were diluted into 300 mL HMI-9 medium containing 5 ng/mL tet. Cells were allowed to recover for 6 h before addition of puromycin and ganciclovir for selection. An aliquot of cells was also taken following transfection and serially diluted in media containing puromycin and ganciclovir to estimate transfection efficiency and library complexity. Two separate transfections of the mutant library into independent I-SceI expressing cell lines were carried out with an efficiency of ~2.3 x 10-3, yielding approximately 100,000 and 120,000 transfectants per library. Following 6 days of selection and growth to a total of >1 x 108 cells, genomic DNA (gDNA) was harvested from 5 x 107 cells per library transfection (>400x of each transfectant) as our input samples. Another 5 x 107 cells were then washed to remove tet and resuspended in 175 mL HMI-9 and grown for 4 days as described above. During growth, the total cell numbers in our BF library pools were always kept above 5 x 107, with density below 2 x 106 cells/mL to maintain library complexity and growth in logarithmic phase. gDNA was harvested from 5 x 107 cells on day 4 following tet withdrawal, reflecting the time-point at which expression of tet-regulated WT B4 is robustly repressed, and growth defects are observed in the parental BF B4 CN cells. gDNA extraction was carried out using the NucleoSpin Blood kit (Macherey-Nagal), eluting in a volume of 100 L. A total of 24 cycles of PCR amplification were used to amplify the mutated domain and add sequencing primer binding sites, indices, and P5/P7 flow cell attachment sites for Illumina sequencing. To ascertain the range of linear amplification for specific primer sets (Supplemental Table 1), real-time PCR using SsoAdvanced Universal SYBR Green Supermix (BioRad) was performed (not shown). The final amplicon size was 423 bp. PCR products were visualized and purified by electrophoresis on FlashGel DNA cassettes (Lonza). PCR amplicon concentrations were determined using a Qubit Fluorometer (ThermoFisher Scientific), and molar concentrations calculated using the final amplicon size. Equimolar amounts of all indexed amplicons were then combined into a single library for analysis on the Illumina MiSeq. All amplicons were sequenced together on a single flow cell to eliminate the possibility of uncontrolled variability between sequencing runs.